This invention relates to the preparation of a DNA sequence which codes for a polypeptide having the biological activity of the enzyme mutarotase.
In addition, the invention relates to recombinant DNA molecules, that is to say cloning and expression vectors, for use in the preparation of a polypeptide having the biological activity of the enzyme mutarotase, and to host organisms transformed with such vectors, for example bacteria, yeasts, other fungi, and animal or human cells.
The invention also relates to the use of the said polypeptide having the biological activity of the enzyme mutarotase for increasing the rate of enzymatic detection reactions or conversions of aldoses.
Mutarotase (aldose 1-epimerase, EC 5.1.3.3) is known to increase the rate of setting up equilibrium between the .alpha.- and .beta.-anomers of aldohexoses, for example, between .alpha.- and .beta.-glucose or .alpha.- and .beta.-galactose. The main use of the enzyme is in analytical biochemistry for increasing the rate of enzymatic detection reactions for aldoses by means of enzymes specific for the .alpha.- or .beta.-form, in which the setting up of the equilibrium between the two anomers is the rate-determining step, for example in determination methods with glucose dehydrogenase, glucose oxidase or galactose dehydrogenase.
Industrial use would be of interest, for example for the glucoamylase/glucose isomerase process, because the enzyme glucoamylase liberates .beta.-glucose which cannot be converted into the .alpha.-form by the glucose isomerase until mutarotation has taken place.
Mutarotase is widespread in nature, occurring in various microorganisms (bacteria, yeasts and filamentous fungi), in plants and in animal tissues.
The only considerable enzyme contents allowing isolation of mutarotase on the industrial scale have to date been found in the kidneys of mammals (cattle, pigs); all the known commercial products are prepared from kidneys. It is known, from Bailey, Meth. Enzymol. 1975, 478, that the content of mutarotase activity per g of fresh weight in bovine kidneys is more than 60 times that in, for example, Escherichia coli. A process for the microbiological preparation of mutarotase from strains of Aspergillus niger was described for the first time in Biochim. Biophys. Acta 662, 285 (1981). According to this, the mutarotase activity obtained from the best strain was 4.4 mU/ml of culture broth, the Michaelis constant being 50 mM and the pH optimum being in the range 5 to 7.
However, using the said microbiological process the enzyme mutarotase can only be obtained in lower yields than with the process for preparation from bovine kidneys. In addition, the properties of the enzyme from Aspergillus niger are unfavorable for setting up the equilibrium within the scope of enzymatic determinations of aldoses.